Rutao Cui

Central Role of p53 in the Suntan Response and Pathologic Hyperpigmentation

UV-induced pigmentation (suntanning) requires induction of alpha-melanocyte-stimulating hormone (alpha-MSH) secretion by keratinocytes. alpha-MSH and other bioactive peptides are cleavage products of pro-opiomelanocortin (POMC). Here we provide biochemical and genetic evidence demonstrating that UV induction of POMC/MSH in skin is directly controlled by p53. Whereas p53 potently stimulates the POMC promoter in response to UV, the absence of p53, as in knockout mice, is associated with absence of the UV-tanning response. The same pathway produces beta-endorphin, another POMC derivative, which potentially contributes to sun-seeking behaviors. Furthermore, several instances of UV-independent pathologic pigmentation are shown to involve p53 mimicking the tanning response. p53 thus functions as a sensor/effector for UV pigmentation, which is a nearly constant environmental exposure. Moreover, this pathway is activated in numerous conditions of pathologic pigmentation and thus mimics the tanning response.

Topical drug rescue strategy and skin protection based on the role of Mc1r in UV-induced tanning

Ultraviolet-light (UV)-induced tanning is defective in numerous ‘fair-skinned’ individuals, many of whom contain functional disruption of the melanocortin 1 receptor (MC1R). Although this suggested a critical role for the MC1R ligand melanocyte stimulating hormone (MSH) in this response, a genetically controlled system has been lacking in which to determine the precise role of MSH–MC1R. Here we show that ultraviolet light potently induces expression of MSH in keratinocytes, but fails to stimulate pigmentation in the absence of functional MC1R in red/blonde-haired Mc1re/e mice. However, pigmentation could be rescued by topical application of the cyclic AMP agonist forskolin, without the need for ultraviolet light, demonstrating that the pigmentation machinery is available despite the absence of functional MC1R. This chemically induced pigmentation was protective against ultraviolet-light-induced cutaneous DNA damage and tumorigenesis when tested in the cancer-prone, xeroderma-pigmentosum-complementation-group-C-deficient genetic background. These data emphasize the essential role of intercellular MSH signalling in the tanning response, and suggest a clinical strategy for topical small-molecule manipulation of pigmentation.

miR-200c Inhibits Melanoma Progression and Drug Resistance through Down-Regulation of Bmi-1

MicroRNAs (miRNAs) are short noncoding RNAs that play crucial roles in tumorigenesis and tumor progression. Melanoma is the most aggressive skin cancer that is resistant or rapidly develops resistance to a variety of chemotherapeutic agents. The role of miRNAs in melanoma progression and drug resistance has not been well studied. Herein, we demonstrate that miR-200c is down-regulated in melanomas (primary and metastatic) compared with melanocytic nevi. Overexpression of miR-200c in melanoma cells resulted in significantly decreased cell proliferation and migratory capacity as well as drug resistance. miR-200c overexpression resulted in significant down-regulation of BMI-1, ABCG2, ABCG5, and MDR1 expression and in a concomitant increase in E-cadherin levels. Knockdown of BMI-1 showed similar effects as miR-200c overexpression in melanoma cells. In addition, miR-200c overexpression significantly inhibited melanoma xenograft growth and metastasis in vivo, and this correlated with diminished expression of BMI-1 and reduced levels of E-cadherin in these tumors. The effects of miR-200c on melanoma cell proliferation and migratory capacity and on self-renewal were rescued by overexpression of Bmi-1, and the reversal of these phenotypes correlated with a reduction in E-cadherin expression and increased levels of ABCG2, ABCG5, and MDR1. Taken together, these findings demonstrate a key role for miR-200c in melanoma progression and drug resistance. These results suggest that miR-200c may represent a critical target for increasing melanoma sensitivity to clinical therapies.

MC1R is a potent regulator of PTEN after UV exposure in melanocytes.

The individuals carrying melanocortin-1 receptor (MC1R) variants, especially those associated with red hair color, fair skin, and poor tanning ability (RHC trait), are more prone to melanoma; however, the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers phosphatase and tensin homolog (PTEN) interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAF(V600E), MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes response to UVB exposure and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis.

Stat3-Targeted Therapies Overcome the Acquired Resistance to Vemurafenib in Melanomas

Vemurafenib (PLX4032), a selective inhibitor of Braf, has been approved by the US Food and Drug Administration for the treatment of unresectable or metastatic melanoma in patients with Braf(V600E) mutations. Many patients treated with vemurafenib initially display dramatic improvement, with decreases in both risk of death and tumor progression. Acquired resistance, however, rapidly arises in previously sensitive cells. We attempted to overcome this resistance by targeting the signal transducer and activator of transcription 3 (STAT3)-paired box homeotic gene 3 (PAX3)-signaling pathway, which is upregulated, owing to fibroblast growth factor 2 (FGF2) secretion or increased kinase activity, with the Braf(V600E) mutation. We found that activation of Stat3 or overexpression of PAX3 induced resistance to vemurafenib in melanoma cells. In addition, PAX3 or Stat3 silencing inhibited the growth of melanoma cells with acquired resistance to vemurafenib. Furthermore, treatment with the Stat3 inhibitor, WP1066, resulted in growth inhibition in both vemurafenib-sensitive and -resistant melanoma cells. Significantly, vemurafenib stimulation induced FGF2 secretion from keratinocytes and fibroblasts, which might uncover, at least in part, the mechanisms underlying targeting Stat3-PAX3 signaling to overcome the acquired resistance to vemurafenib. Our results suggest that Stat3-targeted therapy is a new therapeutic strategy to overcome the acquired resistance to vemurafenib in the treatment of melanoma.

MIR106B and MIR93 Prevent Removal of Bacteria From Epithelial Cells by Disrupting ATG16L1-Mediated Autophagy

BACKGROUND & AIMSnVariants in genes that regulate autophagy have been associated with Crohns disease (CD). Defects in autophagy-mediated removal of pathogenic microbes could contribute to the pathogenesis of CD. We investigated the role of the microRNAs (miRs) MIR106B and MIR93 in induction of autophagy and bacterial clearance in human cell lines and the correlation between MIR106B and autophagy-related gene 16L1 (ATG16L1) expression in tissues from patients with CD.nnnMETHODSnWe studied the ability of MIR106B and MIR93 to regulate ATG transcripts in human cancer cell lines (HCT116, SW480, HeLa, and U2OS) using luciferase report assays and bioinformatics analyses; MIR106B and MIR93 mimics and antagonists were transfected into cells to modify levels of miRs. Cells were infected with LF82, a CD-associated adherent-invasive strain of Escherichia coli, and monitored by confocal microscopy and for colony-forming units. Colon tissues from 41 healthy subjects (controls), 22 patients with active CD, 16 patients with inactive CD, and 7 patients with chronic inflammation were assessed for levels of MIR106B and ATG16L1 by in situ hybridization and immunohistochemistry.nnnRESULTSnSilencing Dicer1, an essential processor of miRs, increased levels of ATG protein and formation of autophagosomes in cells, indicating that miRs regulate autophagy. Luciferase reporter assays indicated that MIR106B and MIR93 targeted ATG16L1 messenger RNA. MIR106B and MIR93 reduced levels of ATG16L1 and autophagy; these increased after expression of ectopic ATG16L1. In contrast, MIR106B and MIR93 antagonists increased formation of autophagosomes. Levels of MIR106B were increased in intestinal epithelia from patients with active CD, whereas levels of ATG16L1 were reduced compared with controls. Levels of c-Myc were also increased in intestinal epithelia of patients with active CD compared with controls. These alterations could impair removal of CD-associated bacteria by autophagy.nnnCONCLUSIONSnIn human cell lines, MIR106B and MIR93 reduce levels of ATG16L1 and autophagy and prevent autophagy-dependent eradication of intracellular bacteria. This process also appears to be altered in colon tissues from patients with active CD.

PGC-1 Coactivators Regulate MITF and the Tanning Response

The production of pigment by melanocytes tans the skin and protects against skin cancers. UV-exposed keratinocytes secrete α-MSH, which then activates melanin formation in melanocytes by inducing the microphthalmia-associated transcription factor (MITF). We show that PPAR-γ coactivator (PGC)-1α and PGC-1β are critical components of this melanogenic system in melanocytes. α-MSH signaling strongly induces PGC-1α expression and stabilizes both PGC-1α and PGC-1β proteins. The PGC-1s in turn activate the MITF promoter, and their expression correlates strongly with that of MITF in human melanoma cell lines and biopsy specimens. Inhibition of PGC-1α and PGC-1β blocks the α-MSH-mediated induction of MITF and melanogenic genes. Conversely, overexpression of PGC-1α induces pigment formation in cell culture and transgenic animals. Finally, polymorphism studies reveal expression quantitative trait loci in the PGC-1β gene that correlate with tanning ability and protection from melanoma in humans. These data identify PGC-1 coactivators as regulators of human tanning.

Family Members p53 and p73 Act Together in Chromatin Modification and Direct Repression of α-Fetoprotein Transcription

Aberrant expression of the α-fetoprotein (AFP) gene is a diagnostic tumor marker of hepatocellular carcinoma. We find that AFP gene expression is repressed by the TP53 family member p73 during normal hepatic development and when p73α or p73β is introduced into cultured hepatoma cells that express AFP. Transient co-transfection of p53 family members showed that p53 and transactivating (TA)-p73, but not TA-p63, repress endogenous AFP transcription additively or independently. p53-independent functions of p73 are further supported by delayed, p73-associated compensation of AFP repression during development of the p53-null mouse. Chromatin immunoprecipitation assays of normal and p53-null mouse liver tissue showed that TA-p73 binds at a previously identified p53 repressor site (-860/-830) within the distal promoter of AFP at a level equivalent to p53 in wild type liver, with increased binding of TA-p73 to chromatin in the absence of p53. Sequential chromatin immunoprecipitation analyses revealed that TA-p73 and p53 bind simultaneously to their shared regulatory site in wild type liver. Like the founding family member p53, TA-p73 represses AFP expression by chromatin structure alteration, targeting reduction of acetylated histone H3 lysine 9 and increased dimethylated histone H3 lysine 9 levels. However, chromatin-bound TA-p73 is associated with elevated di- and tri-methylated histone H3 lysine 4 levels in p53-null liver and hepatoma cells, concomitant with a reduced ability to repress transcription compared with p53.

Central role of cellular senescence in TSLP-induced airway remodeling in asthma.

Background Airway remodeling is a repair process that occurs after injury resulting in increased airway hyper-responsiveness in asthma. Thymic stromal lymphopoietin (TSLP), a vital cytokine, plays a critical role in orchestrating, perpetuating and amplifying the inflammatory response in asthma. TSLP is also a critical factor in airway remodeling in asthma. Objectives To examine the role of TSLP-induced cellular senescence in airway remodeling of asthma in vitro and in vivo. Methods Cellular senescence and airway remodeling were examined in lung specimens from patients with asthma using immunohischemical analysis. Both small molecule and shRNA approaches that target the senescent signaling pathways were used to explore the role of cellular senescence in TSLP-induced airway remodeling in vitro. Senescence-Associated β-galactosidase (SA-β-Gal) staining, and BrdU assays were used to detect cellular senescence. In addition, the Stat3-targeted inhibitor, WP1066, was evaluated in an asthma mouse model to determine if inhibiting cellular senescence influences airway remodeling in asthma. Results Activation of cellular senescence as evidenced by checkpoint activation and cell cycle arrest was detected in airway epithelia samples from patients with asthma. Furthermore, TSLP-induced cellular senescence was required for airway remodeling in vitro. In addition, a mouse asthma model indicates that inhibiting cellular senescence blocks airway remodeling and relieves airway resistance. Conclusion TSLP stimulation can induce cellular senescence during airway remodeling in asthma. Inhibiting the signaling pathways of cellular senescence overcomes TSLP-induced airway remodeling.

FGF2 regulates melanocytes viability through the STAT3-transactivated PAX3 transcription

PAX3 (paired box 3) is known to have an important role in melanocyte development through modulation of microphthalmia-associated transcription factor transcription. Here we found that PAX3 transcriptional activity could be regulated through FGF2 (basic fibroblast growth factor)-STAT3 (signal transducer and activator of transcription 3) signaling in the pigment cells. To study its function in vivo, we have generated a transgenic mouse model expressing PAX3 driven by tyrosinase promoter in a tissue-specific fashion. These animals exhibit hyperpigmentation in the epidermis, evident in the skin color of their ears and tails. We showed that the darker skin color results from both increased melanocyte numbers and melanin synthesis. Together, our study delineated a novel pathway in the melanocyte lineage, linking FGF2-STAT3 signaling to increased PAX3 transcription. Moreover, our results suggest that this pathway might contribute to the regulation of melanocyte numbers and melanin levels, and thereby provide an alternative strategy to induce pigmentation.